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Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila.

Sun NK, Sun CL, Lin CH, Pai LM, Chao CC - J. Biomed. Sci. (2010)

Bottom Line: Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP.On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger.Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Chang Gung University, Gueishan, Taoyuan 333, Taiwan.

ABSTRACT

Background: We observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process.

Methods: We explored the role of DDB2 in mediating UV resistance in both human cells and Drosophila. To do so, DDB2 was overexpressed by using a full-length open reading frame cDNA. Conversely, DDB2 and cFLIP were suppressed by using antisense oligonucleotides. Cell survival was measured using a colony forming assay. Apoptosis was monitored by examination of nuclear morphology, as well as by flow cytometry and Western blot analyses. A transcription reporter assay was also used to assess transcription of cFLIP.

Results: We first observed that the cFLIP protein was upregulated in UV-resistant HeLa cells. In addition, the cFLIP protein could be induced by stable expression of DDB2 in these cells. Notably, the anti-apoptotic effect of DDB2 against UV irradiation was largely attenuated by knockdown of cFLIP with antisense oligonucleotides in HeLa cells. Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP. Interestingly, ectopic expression of human DDB2 in Drosophila dramatically inhibited UV-induced fly death compared to control GFP expression. On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger.

Conclusion: Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner. In addition, the protective role of DDB2 against UV irradiation was found to be conserved in divergent living organisms such as human and Drosophila. In addition, UV irradiation may activate a cFLIP-regulated apoptotic pathway in certain cells.

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Stimulation of cFLIP expression and attenuation of UV sensitivity by forced expression of DDB2. (A) Overexpression of cFLIP in HR3 cells. The plotted values represent means ± S.D. of three experiments (right panel). (B) Stimulation of cFLIP expression by forced expression of DDB2. Whole cell extracts of HR18 cells, treated as indicated, were subjected to immunoblot analysis with specific antibodies. (C) Cell sensitivity to UV after Ad-DDB2 virus infection. The plotted values represent means ± S.D. of three experiments. ** Significant difference against control (p < 0.05).
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Figure 3: Stimulation of cFLIP expression and attenuation of UV sensitivity by forced expression of DDB2. (A) Overexpression of cFLIP in HR3 cells. The plotted values represent means ± S.D. of three experiments (right panel). (B) Stimulation of cFLIP expression by forced expression of DDB2. Whole cell extracts of HR18 cells, treated as indicated, were subjected to immunoblot analysis with specific antibodies. (C) Cell sensitivity to UV after Ad-DDB2 virus infection. The plotted values represent means ± S.D. of three experiments. ** Significant difference against control (p < 0.05).

Mentions: The protective effect of DDB2 against UV irradiation may be associated with various regulators of apoptosis. To assess this possibility, we examined the level of cFLIP, Bcl-2, and Bcl-xL in UV-resistant cells. The level of cFLIP protein was increased in HR3 cells compared to control HeLa cells (Fig. 3A). On the other hand, HR18 cells, which express a low level of DDB2, showed low cFLIP compared to control cells (Fig. 3A). Notably, we found that overexpression of DDB2 using an adenovirus system increased the level of cFLIP in HR18 cells (Fig. 3B). In this case, increased expression of DDB2 was noticed 24 hrs following virus infection, whereas the level of cFLIP increased only 36 hrs following virus infection (Fig. 3B). Overexpression of control Gal did not influence the level of DDB2 or cFLIP compared to mock-treated cells (Fig. 3B). The stimulation of cFLIP following overexpression of DDB2 was also detected in HeLa cells (data not shown). To verify whether DDB2 could enhance resistance to UV, we overexpressed DDB2 in HR18 cells and monitored apoptosis following UV irradiation. Overexpression of DDB2 was shown to protect against UV irradiation in a time-dependent manner (Fig. 3C). UV resistance correlated with the level of cFLIP protein in this case since UV resistance was maximum at 36 and 72 hours following virus infection (Figs. 3B and 3C). These results indicate that UV resistance may be associated with increased levels of DDB2 and cFLIP.


Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila.

Sun NK, Sun CL, Lin CH, Pai LM, Chao CC - J. Biomed. Sci. (2010)

Stimulation of cFLIP expression and attenuation of UV sensitivity by forced expression of DDB2. (A) Overexpression of cFLIP in HR3 cells. The plotted values represent means ± S.D. of three experiments (right panel). (B) Stimulation of cFLIP expression by forced expression of DDB2. Whole cell extracts of HR18 cells, treated as indicated, were subjected to immunoblot analysis with specific antibodies. (C) Cell sensitivity to UV after Ad-DDB2 virus infection. The plotted values represent means ± S.D. of three experiments. ** Significant difference against control (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864207&req=5

Figure 3: Stimulation of cFLIP expression and attenuation of UV sensitivity by forced expression of DDB2. (A) Overexpression of cFLIP in HR3 cells. The plotted values represent means ± S.D. of three experiments (right panel). (B) Stimulation of cFLIP expression by forced expression of DDB2. Whole cell extracts of HR18 cells, treated as indicated, were subjected to immunoblot analysis with specific antibodies. (C) Cell sensitivity to UV after Ad-DDB2 virus infection. The plotted values represent means ± S.D. of three experiments. ** Significant difference against control (p < 0.05).
Mentions: The protective effect of DDB2 against UV irradiation may be associated with various regulators of apoptosis. To assess this possibility, we examined the level of cFLIP, Bcl-2, and Bcl-xL in UV-resistant cells. The level of cFLIP protein was increased in HR3 cells compared to control HeLa cells (Fig. 3A). On the other hand, HR18 cells, which express a low level of DDB2, showed low cFLIP compared to control cells (Fig. 3A). Notably, we found that overexpression of DDB2 using an adenovirus system increased the level of cFLIP in HR18 cells (Fig. 3B). In this case, increased expression of DDB2 was noticed 24 hrs following virus infection, whereas the level of cFLIP increased only 36 hrs following virus infection (Fig. 3B). Overexpression of control Gal did not influence the level of DDB2 or cFLIP compared to mock-treated cells (Fig. 3B). The stimulation of cFLIP following overexpression of DDB2 was also detected in HeLa cells (data not shown). To verify whether DDB2 could enhance resistance to UV, we overexpressed DDB2 in HR18 cells and monitored apoptosis following UV irradiation. Overexpression of DDB2 was shown to protect against UV irradiation in a time-dependent manner (Fig. 3C). UV resistance correlated with the level of cFLIP protein in this case since UV resistance was maximum at 36 and 72 hours following virus infection (Figs. 3B and 3C). These results indicate that UV resistance may be associated with increased levels of DDB2 and cFLIP.

Bottom Line: Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP.On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger.Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Chang Gung University, Gueishan, Taoyuan 333, Taiwan.

ABSTRACT

Background: We observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process.

Methods: We explored the role of DDB2 in mediating UV resistance in both human cells and Drosophila. To do so, DDB2 was overexpressed by using a full-length open reading frame cDNA. Conversely, DDB2 and cFLIP were suppressed by using antisense oligonucleotides. Cell survival was measured using a colony forming assay. Apoptosis was monitored by examination of nuclear morphology, as well as by flow cytometry and Western blot analyses. A transcription reporter assay was also used to assess transcription of cFLIP.

Results: We first observed that the cFLIP protein was upregulated in UV-resistant HeLa cells. In addition, the cFLIP protein could be induced by stable expression of DDB2 in these cells. Notably, the anti-apoptotic effect of DDB2 against UV irradiation was largely attenuated by knockdown of cFLIP with antisense oligonucleotides in HeLa cells. Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP. Interestingly, ectopic expression of human DDB2 in Drosophila dramatically inhibited UV-induced fly death compared to control GFP expression. On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger.

Conclusion: Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner. In addition, the protective role of DDB2 against UV irradiation was found to be conserved in divergent living organisms such as human and Drosophila. In addition, UV irradiation may activate a cFLIP-regulated apoptotic pathway in certain cells.

Show MeSH
Related in: MedlinePlus